PROJECT SUMMARY/ABSTRACT One of the most important limitations to modern medicine is the substantial and often unpredictable variation between patients in their response to treatment. It is now well established that variations in the human genome, in particular the enzymes and transporters expressed in the intestine and liver, have a major impact on drug levels in circulation. But these studies ignore the genetic variation in our ?second genome? ? that of the trillions of microorganisms that thrive in and on the human body (the microbiome). To date, studies have shown that >40 drugs can be metabolized by the gut microbiome spanning many of the most intractable chronic diseases: cancer, heart disease, and inflammatory bowel disease. Yet very little progress has been made to translate these findings due to a lack of knowledge about the microbial enzymes responsible and how environmental factors like dietary intake shape their activity. As an initial proof-of-principle, we chose to focus on the cardiac drug digoxin, prescribed for heart failure and irregular heartbeat. Digoxin is an ideal test case for multiple reasons: (i) a single reaction, uniquely catalyzed by gut bacteria, inactivates the drug; (ii) minor changes to drug levels are clinically relevant due to its narrow therapeutic range; and (iii) Eggerthella lenta is the only gut bacterium that has been shown to catalyze this reaction. We recently identified the bacterial enzymes responsible for digoxin reduction (Haiser et al., Science 2013), providing the first mechanistic explanation for how inter-individual differences in the gut microbiome contribute to variations in drug levels. Our preliminary results suggest that two factors are important in controlling the inactivation of digoxin by gut bacteria: strain-level variation in E. lenta population and host dietary intake. We will systematically dissect these two factors, determining how and why they impact drug levels. These studies will provide basic biological insights into a poorly studied but clinically-relevant bacterial species, while moving towards our long-term goal of optimizing treatment outcomes by pairing microbiome-based diagnostic tests and nutritional guidelines.